Alcohol-exposed Rats Research Articles

PROTECTIVE EFFECT OF THE CALCIUM CHANNEL BLOCKER, NIFEDIPINE, AGAINST ALCOHOL WITHDRAWAL-INDUCED ANXIETY IN RATS

The objective of the present study was to measure anxiety as a sign of alcohol withdrawal in an animal model of anxiety and to assess the protective effect of the calcium channel blocker, nifedipine. against alcohol-withdrawal -induced anxiety in male rats. Fifty-six adult male albino rats were used in the current study. Animals were randomly divided into t10 groups. normal rats and alcohol- exposed rats. The normal animals were given free access to tap water: the normal group staved alcohol naive for the remainder period of the study. The animals of the alcohol-exposed group were given free access to ethanol solution (10% v/v) in their home cages. Rats were given access to ethanol in their home cages for three cycles of 5 consecutive days, interspersed by two days of tap Water. Each group, normal or alcohol-exposed group, was further randomly subdivided into 4 subgroups. 7 rats each: the animals of these subgroups received the following treatments: Normal saline (NACT 0.9%). Diazepam at a dose of 0.5mg/kg. i.p. 30-minutes before anxiety test, Nifedipine at doses of 10mg/kg. i.p. 30-minutes before anxiety test. And Nifedipine. 5mg/kg. i.p. daily for 5 days a Week for 3 weeks along with ethanol exposure. Test of anxiety was conducted after 3 cycles of tap water or ethanol exposure. Tap water on ethanol solution was removed from all animal cages at 4:00p.m the day before anxiety testing to induce water-deprivation in normal animals and alcohol-withdrawal in alcohol-exposed rats. respectively. The present results show that alcohol exposure was effective in producing anxiety as a sign of alcohol-withdrawal in rats. Treatment with the lower dose of nifedipine (5mg/kg). for three weeks along with alcohol exposure, was more effective in alleviating the -induced anxiety as compared to the acute effect of the higher dose (nifedipine, 10mg/kg). Diazepam induced a comparable anxiolytic effect. It could be concluded that calcium channel blockers may offer a possible substitute for benzodiazepine anxiolytics to treat alcohol-withdrawal symptoms because they do not induce physical dependence.

Cognitive impairment and increased brain neurosteroids in adult rats perinatally exposed to low millimolar blood alcohol concentrations

Epidemiological evidence suggests that adolescents and adults perinatally exposed to alcohol, even at low doses, show high prevalence of cognitive impairment and social behavior deficits, which may be in part related to alcohol-induced changes of the gamma-aminobutyric acid (GABA)ergic neurotransmission. The endogenous neurosteroid 3alpha-hydroxy,5alpha-pregnan-20-one (3alpha,5alpha-tetrahydroprogesterone/3alpha,5alpha-THP), a potent positive allosteric modulator of GABA(A) receptor function, is implicated in the physiological tuning of GABA-mediated fast inhibition and in various alcohol's actions in the brain. This study was undertaken to determine whether perinatal exposure to low millimolar blood alcohol concentrations alters cognitive skills (social discrimination and inhibitory avoidance tests), emotional reactivity (elevated plus maze test), and neurosteroid content in brain cortex and hippocampus of adult male offspring. Dams had access to a 3% alcohol solution or to an equicaloric sucrose solution from gestational day 15 to postnatal day 9. Eighty-day old alcohol-exposed male offspring exhibited impaired social recognition memory, but unchanged inhibitory avoidance performance and normal behavior on the elevated-plus maze. The concentrations of 3alpha,5alpha-THP and its precursor progesterone were more than doubled in brain cortex and hippocampus of alcohol-exposed rats, whereas in plasma only progesterone was increased. Thus, exposure to low millimolar blood alcohol concentrations has a long-lasting impact on the developing brain as it causes an impairment of social recognition as well as an increase of brain neurosteroid content in mature animals. The latter may be consequent to altered expression/activity of brain steroidogenic enzymes, as reflected by the enduring increase of the GABA(A) receptor-active neurosteroid 3alpha,5alpha-THP in brain cortex and hippocampus, but not in plasma. It is speculated that, by inducing a greater amplification of GABA(A) receptor function, the elevation of 3alpha,5alpha-THP brain content contributes to the cognitive impairment exhibited by adult alcohol-exposed offspring.

Effects of chronic alcohol exposure on dopamine uptake in rat nucleus accumbens and caudate putamen

Existing data strongly suggest that alcohol affects dopamine (DA) neurotransmission in the brain. However, many questions remain about the effects of alcohol on the delicate equilibrium between such neurochemical processes as DA release and uptake. Dysregulation of these processes in the mesolimbic and nigrostriatal systems after chronic alcohol ingestion could be a neuroadaptation contributing to dependence. In the present study, we have employed an alcohol vapor inhalation model to characterize the effects of chronic alcohol exposure on DA dynamics in rat nucleus accumbens (NAc) and caudate putamen (CP) using fast-scan cyclic voltammetry (FSCV) in brain slices. This method provides a unique view of real-time, spatially resolved changes in DA concentration. We found that chronic alcohol exposure enhanced DA uptake rates in rat NAc and CP. These changes would have the effect of down-regulating extracellular DA levels, presumably a compensatory effect related to increased DA release by repeated alcohol exposure. The sensitivity of terminal release-regulating DA autoreceptors was not different in alcohol-exposed rats compared with alcohol-naïve animals. The DA uptake changes after chronic alcohol exposure documented here using FSCV may be associated with a compensatory response of the DA system aimed at decreasing DA signaling. Alterations in autoreceptor function may require relatively long lasting alcohol exposure.

Interaction of Thiamine Deficiency and Voluntary Alcohol Consumption Disrupts Rat Corpus Callosum Ultrastructure

The relative roles of alcohol and thiamine deficiency in causing brain damage remain controversial in alcoholics without the Wernicke-Korsakoff syndrome. Experimental control over alcohol consumption and diet are impossible in humans but can be accomplished in animal models. This experiment was designed to differentiate the separate and combined effects on the macro- and ultrastructure of the corpus callosum of thiamine deficiency and voluntary alcohol consumption. Adult male alcohol-preferring (P) rats (9 chronically alcohol-exposed and 9 water controls) received a thiamine-deficient diet for 2 weeks. There were four groups: five rats previously exposed to alcohol were treated with pyrithiamine (a thiamine phosphorylation inhibitor); five rats never exposed to alcohol were treated with pyrithiamine; four alcohol-exposed rats were treated with thiamine; and four rats never exposed to alcohol were treated with thiamine. On day 14, thiamine was restored in all 18 rats; 2 weeks later the 10 pyrithiamine-treated rats received intraperitoneal thiamine. The rats were perfused 61 days post-pyrithiamine treatment at age 598 days. Brains were dissected and weight and volumes were calculated. Sagittal sections were stained to measure white matter structures. The corpus callosum was examined using transmission electron microscopy to determine density of myelinated fibers, fiber diameter, and myelin thickness. The corpus callosum in the alcohol/pyrithiamine group was significantly thinner, had greater fiber density, higher percentage of small fibers, and myelin thinning than in the alcohol/thiamine and water/thiamine groups. Several measures showed a graded effect, where the alcohol/pyrithiamine group had greater pathology than the water/pyrithiamine group, which had greater pathology than the two thiamine-replete groups. Across all 16 rats, thinner myelin sheaths correlated with higher percentage of small fibers. Myelin thickness and axon diameter together accounted for 71% of the variance associated with percentage of small fibers. Significant abnormalities in the alcohol/pyrithiamine group and lack of abnormality in the alcohol-exposed/thiamine-replete group indicate that thiamine deficiency caused white matter damage. The graded abnormalities across the dually to singly treated animals support a compounding effect of alcohol exposure and thiamine depletion, and indicate the potential for interaction between alcohol and thiamine deficiency in human alcohol-related brain damage.

MODULATION OF THE INTERLEUKIN-13 SIGNALING PATHWAY IN THE RAT ALCOHOLIC LUNG.

Chronic alcohol exposure in rats induces oxidative stress in the lung via the angiotensin II pathway and elicits an increase in expression of the profibrotic mediator, transforming growth factor β1 (TGF-β1). In addition, fibronectin expression is increased in the lungs of alcohol-fed rats. These data strongly suggest that profibrotic mechanisms are activated in response to chronic alcohol exposure, and if true, this would suggest that alcohol abuse might increase the risk of fibrotic lung diseases. Consistent with this hypothesis, we have recently determined that alcohol ingestion increases TGF-β1 expression and collagen deposition in an experimental model of obliterative bronchiolitis (OB) following tracheal transplantation in rats. Another profibrotic cytokine, interleukin-13 (IL-13), has been shown to be an important mediator of tissue remodeling/fibrosis in the lung and may act upstream of the TGF-β1 pathway. We hypothesized that alterations in the expression of IL-13 or its receptors could contribute to alcohol-mediated amplification of profibrotic mediators in the alcoholic lung. To test this hypothesis, we analyzed whole lungs and type II alveolar epithelial cells isolated from alcohol-fed rats, as well as mouse lung fibroblasts exposed to alcohol in vitro, for various components of the IL-13 signaling pathway. Gene expression of IL-13, along with the α1 and α2 subunits of its receptor (IL-13Rα1 and IL-13Rα2), was examined by assessing mRNA levels by RT-PCR. In parallel, IL-13Rα2 protein expression was determined by immunostaining. We found that IL-13Rα2 protein was expressed in the whole lung, lung fibroblasts, and type II epithelial cells. RT-PCR analyses indicated that whole lung expresses all three components of the IL-13 signaling pathway. Although lungs from alcohol-fed rats displayed decreased IL-13 mRNA expression, IL-13Rα1 expression was increased, and a marked elevation in IL-13Rα1mRNA expression was observed in alcohol-treated primary lung fibroblasts. Associated with the increase in IL-13Rα1 mRNA in lung fibroblasts was an increase in α-smooth muscle actin protein expression, suggesting fibroblast transdifferentiation into myofibroblasts, which is a cardinal feature of fibrotic diseases such as idiopathic pulmonary fibrosis and bronchiolitis obliterans. These data suggest that IL-13 and its receptors play a role in alcohol-mediated activation of profibrotic mechanisms. In particular, we speculate that alcohol abuse increases IL-13Rα1 expression, which could heighten the response of cells in the lung to IL-13, thereby amplifying TGF-β1-induced fibroblast activation and subsequent tissue remodeling.

Neuropatía óptica inducida por exposición prenatal a drogas o alcohol

The main aim of this work was to analyse the cellular and molecular mechanisms involved in retinal and optic nerve development, and the consequences of methamphetamine "ice" (MA) or alcohol (EtOH) abuse during pregnancy on the developing visual system. Wistar rats were exposed to MA or EtOH during gestation and lactation and their offspring studied. Control isocaloric rats were maintained in parallel. The eyes and optic nerves from pups (at 7, 14 and 21 postnatal days) were processed using morphologic, morphometric and western blot approaches using antibodies against glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and neurofilament protein (NFP). Statistically significant differences were observed between the methamphetamine-exposed and the alcohol-exposed rats, as compared to the controls. The optic nerve cross-sectional area was smaller in the drug or alcohol-exposed animals. The expression of developmental protein markers (GFAP and MBP) in the retina and optic nerve displayed striking alterations related to drug or alcohol abuse during gestation and lactation. Psychostimulant and alcohol exposure alters the development of the retina and optic nerve.

Memory and Perseveration on a Win-Stay, Lose-Shift Task in Rats Exposed Neonatally to Alcohol

It is important to understand the relationship between perseverative responding resulting from perinatal exposure to alcohol and potential underlying causes, including attention, memory, or response-inhibition problems. The present study was designed to examine the relationship between perseveration and memory. Rats exposed neonatally to 6 g/kg/day alcohol from postnatal day (PD) 4 through PD 9 using an artificial rearing technique (n = 8) were compared with an artificially reared gastrostomy control group (n = 8) and a suckle control group (n = 8). Activity levels were assessed from PD 18-21. Beginning on PD 45, subjects were deprived of food and responded for food on a two-lever win-stay, lose-shift task in which reinforcement probability was a function of reinforcement delivery on the previous trial. If reinforcement was delivered, only a response on the same lever (stay) was reinforced. If reinforcement was not delivered, only a response on the opposite lever (shift) was reinforced. Effective responding depended on subjects remembering whether a reinforcer was delivered on the preceding trial. The intertrial interval varied across conditions (5 seconds or 60 seconds). Alcohol-exposed rats showed increased activity during activity testing but did not differ from controls on win-stay, lose-shift accuracy. All groups showed a performance decrease at longer intertrial intervals. Alcohol-exposed rats showed increased lever pressing during the intertrial interval compared with suckle control rats but not with gastrostomy control rats. Choice behavior was comparable for all groups on the win-stay, lose-shift task, indicating that memory, as assessed by this task, was not differentially affected by alcohol exposure. Alcohol-exposed rats responded more during the intertrial interval compared with suckle controls, suggesting increased activity without increased response inhibition. The win-stay, lose-shift procedure is a potentially useful tool for separating simple activity level effects, memory-related effects, and response-inhibition effects. This study also highlights the need for additional research describing the relationship between perseverative responding and underlying mechanisms.

Development and Resolution of Brain Lesions Caused by Pyrithiamine- and Dietary-Induced Thiamine Deficiency and Alcohol Exposure in the Alcohol-Preferring Rat: A Longitudinal Magnetic Resonance Imaging and Spectroscopy Study

Wernicke's encephalopathy (WE) is characterized by lesions in thalamus, hypothalamus (including mammillary nuclei), and inferior colliculi, results in serious disabilities, has an etiology of thiamine deficiency, is treatable with thiamine, and occurs most commonly with alcoholism. Despite decades of study, whether alcohol exposure exacerbates the neuropathology or retards its resolution remains controversial. To examine patterns of brain damage and recovery resulting from thiamine deprivation with and without alcohol exposure, we conducted in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) at 3 T in alcohol-preferring (P) rats, which had voluntarily consumed large amounts of alcohol before thiamine manipulation. A total of 18 adult male P rats (nine alcohol-exposed) received a thiamine-deficient diet for 2 weeks: 10 (five alcohol-exposed) received intraperitoneal (i.p.) pyrithiamine (PT) and eight (four alcohol-exposed) received i.p. thiamine supplementation. Neurological signs developed by day 14. Rats were scanned before thiamine depletion and 18 and 35 days after thiamine repletion. Two-dimensional J-resolved MRS single-voxel spectra with water reference were collected in a voxel subtending the thalamus; metabolite quantification was corrected for voxel tissue content. MRI identified significant enlargement of dorsal ventricles and increase in signal intensities in thalamus, inferior colliculi, and mammillary nuclei of PT compared with thiamine-treated (TT) groups from MRI 1-2, followed by significant normalization from MRI 2-3 in thalamus and colliculi, but not mammillary nuclei and lateral ventricles. Voxel-by-voxel analysis revealed additional hyperintense signal clusters in the dorsal and ventral hippocampus and enlargement of the fourth ventricle. MRS showed a significant decline and then partial recovery in thalamic N-acetylaspartate, a marker of neuronal integrity, in PT compared with TT rats, with no change detected in creatine, choline, or glutamate. PT rats with prior alcohol exposure exhibited attenuated recovery in the thalamus and arrested growth of the corpus callosum; further, two of the five alcohol-exposed PT rats died prematurely. Parenchymal and ventricular changes with thiamine manipulation concur with human radiological signs of WE. The enduring macrostructural and neurochemical abnormalities involving critical nodes of Papez circuit carry liabilities for development of amnesia and incomplete recovery from other cognitive and motor functions subserved by the affected neural systems.

Prenatal alcohol exposure and early postnatal changes in the developing nerve-muscle system

Extensive research on prenatal alcohol exposure has proven the potent teratogenicity of this substance of abuse. Children born to alcoholic mothers are often diagnosed with fetal alcohol syndrome (FAS). Those afflicted with FAS often have muscle weakness, muscle wasting, and atrophy. This study assessed the effects of prenatal alcohol exposure on the developing rat neuromuscular system. Pregnant Sprague-Dawley rats were injected intraperitoneally with 1.0 ml of 20% ethyl alcohol/100 gm body weight. Unexposed rats served as controls. The offspring were killed 2, 3, 4, and 5 weeks after birth, and their body weights were recorded. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were recovered and weighed. The TA muscles were histochemically stained by silver cholinesterase in order to study the pattern of innervation. The EDL muscles were processed and stained by hematoxylin-eosin. The number and size of the EDL muscle fibers was quantified. The sciatic nerve was also removed and stained by Swank and Davenport's method to demonstrate the myelin pattern. Assessment at the neuromuscular junction showed a higher proportion of endplates polyneuronally innervated in the alcohol-exposed rats. The muscle weights, as well as the number and size of the muscle fibers, were significantly reduced in these animals. A light-microscopy examination of the nerve sections revealed alterations in the connectivity of myelin. The finding that a higher proportion of endplates were polyneuronally innervated in the alcohol-exposed rats indicates that the maturation process of the neuromuscular system was delayed, thus confirming the deleterious effects of alcohol on growth and maturation of the nerve-muscle system.

Comparison of the impact of melatonin on chronic ethanol‐induced learning and memory impairment between young and aged rats

Chronic alcohol exposure causes functional and structural changes in nervous system which have all been associated with learning and memory impairments. Furthermore, alcohol consumption has been shown to alter the pattern of neural cell adhesion molecules (NCAM) which are involved in memory processes. In the current work, we investigated the effects of melatonin on learning and memory deficits induced by alcohol exposure in young and aged rats. A group of young rats (3 months old) were administered ethanol for 45 days and half of them were co-treated with melatonin. Similar treatments were performed in the aged (19 months old) rats. Morris water maze test and passive avoidance task were used to assess cognitive performance. Lipid peroxidation (LPO) and glutathione (GSH) levels were determined to characterize the level of oxidative stress in the hippocampus and cortex. NCAM levels were determined by Western blotting in the hippocampal homogenates. There was a significant elevation in LPO levels and a reduction in GSH levels in aged and alcohol-exposed rats. Furthermore, both young and aged rats displayed some cognitive impairment when given with alcohol for 45 days. Co-administration of melatonin with ethanol significantly reduced LPO and elevated GSH levels while improving the learning and memory deficits induced by ethanol; the aged rats exhibited a greater response to melatonin supplementation. Moreover, melatonin modulated NCAM expression in hippocampus. Present findings indicate that exposure to ethanol induces learning and memory deficits probably by generating reactive oxygen species and downregulating NCAM 180 in hippocampus of aged rats. Melatonin improves learning and memory deficits and the behavioral responses of rats to melatonin supplementation are age dependent.

Formation of acetaldehyde adducts of glutathione S-transferase A3 in the liver of rats administered alcohol chronically

Hepatic tissue damage induced by chronic exposure to alcohol is mediated through acetaldehyde and associated with reactive oxygen species, which impair cellular defense mechanisms. Because glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics and reactive oxygen species, the current study was undertaken to test the effect of alcohol administration on structural and functional characteristics of rat (r) liver Alpha class rGSTs. Western blot analysis revealed an appreciable change in the expression of rGSTA3 subunit levels, whereas no change was observed in activity after chronic alcohol treatment. Reverse-phase high performance liquid chromatographic analysis of rat liver GSTs that were affinity purified with glutathione showed a 1.07-fold increase in rGSTA3 subunit levels in rats treated with alcohol chronically. In addition, liquid chromatographic–electrospray ionization mass spectrometric analysis of GSTs that were affinity purified with glutathione showed the formation of acetaldehyde adducts to the rGSTA3 subunit. Given the abundant expression of rGSTA3 subunit and acetaldehyde adduct formation, results of the current study support the suggestion that modification of rGSTA3 subunit, and thus its impaired function, in alcohol-exposed rats may contribute to the progression of alcohol-induced liver damage.

In utero alcohol exposure increases mammary tumorigenesis in rats.

Findings in humans and animal models suggest that in utero hormonal and dietary exposures increase later breast cancer risk. Since alcohol intake by adult women consistently increases their breast cancer risk, we wondered whether maternal alcohol consumption during pregnancy increases female offspring's mammary tumorigenesis. In our study, pregnant female rats were pair-fed isocaloric diets containing either 0 (control), 16 or 25 g alcohol kg−1 feed between days 7 and 19 of gestation. These alcohol exposures generate blood alcohol levels that correspond to low and moderate alcohol consumption and are lower than those that induce foetal alcohol syndrome. Serum oestradiol levels were elevated in pregnant rats exposed to alcohol (P<0.003). When adult, female offspring of alcohol-exposed dams developed significantly more 7,12-dimethylbenz[a]anthracene -induced mammary tumours, compared to the controls (tumour multiplicity; mean±s.e.m., controls: 2.0±0.3, 16 g alcohol: 2.7±0.4 and 25 g alcohol: 3.7±0.4; P<0.006). In addition, the mammary epithelial tree of the alcohol-exposed offspring was denser (P<0.004) and contained more structures that are susceptible for the initiation of breast cancer (P<0.001). Immunohistochemical assessment indicated that the mammary glands of 22-week-old in utero alcohol-exposed rats contained elevated levels of oestrogen receptor-α (P<0.04) that is consistent with the changes in mammary gland morphology. In summary, maternal alcohol intake during pregnancy increases female offspring's mammary tumorigenesis, perhaps by programming the foetal mammary gland to exhibit persistent alterations in morphology and gene expression. It remains to be determined whether an increase in pregnancy oestradiol levels mediated alcohol's effects on offspring's mammary tumorigenesis.

Perinatal choline supplementation does not mitigate motor coordination deficits associated with neonatal alcohol exposure in rats

Prenatal alcohol exposure can disrupt brain development, leading to a variety of behavioral alterations including learning deficits, hyperactivity, and motor dysfunction. We have been investigating the possibility that perinatal choline supplementation may effectively reduce the severity of alcohol's adverse effects on behavioral development. We previously reported that perinatal choline supplementation can ameliorate alcohol-induced learning deficits and hyperactivity in rats exposed to alcohol during development. The present study examined whether perinatal choline supplementation could also reduce the severity of motor deficits induced by alcohol exposure during the third trimester equivalent brain growth spurt. Male neonatal rats were assigned to one of three treatment groups. One group was exposed to alcohol (6.6 g/kg/day) from postnatal days (PD) 4 to 9 via an artificial rearing procedure. Artificially and normally reared control groups were included. One half of subjects from each treatment received daily subcutaneous injections of a choline chloride solution from PD 4 to 30, whereas the other half received saline vehicle injections. On PD 35–37, subjects were tested on a parallel bar motor task, which requires both balance and fine motor coordination. Ethanol-exposed subjects exhibited significant motor impairments compared to both control groups whose performance did not differ significantly from one another. Perinatal choline treatment did not affect motor performance in either ethanol or control subjects. These data indicate that the beneficial effects of perinatal choline supplementation in ethanol-treated subjects are task specific and suggest that choline is more effective in mitigating cognitive deficits compared to motor deficits associated with developmental alcohol exposure.

Perinatal choline supplementation attenuates behavioral alterations associated with neonatal alcohol exposure in rats

Children exposed to alcohol prenatally suffer from a variety of behavioral alterations, including hyperactivity and learning deficits. Given that women continue to drink alcohol during pregnancy, it is critical that effective interventions and treatments be identified. Previously, we reported that early postnatal choline supplementation can reduce the severity of learning deficits in rats exposed to alcohol prenatally. The present study examined whether choline supplementation can reduce the severity of behavioral alterations associated with alcohol exposure during the third trimester equivalent brain growth spurt. Male neonatal rats were assigned to one of three treatment groups. One group was exposed to alcohol (6.6 g/kg/day) from postnatal days (PD) 4–9 via an artificial rearing procedure. Artificially reared and normally reared control groups were included. One half of subjects from each treatment received daily subcutaneous injections of a choline chloride solution from PD 4–30, whereas the other half received saline vehicle injections. On PD 31–34, after choline treatment was complete, activity level was monitored and, on PD 40–42, subjects were tested on a serial spatial discrimination reversal learning task. Subjects exposed to alcohol were significantly hyperactive compared to controls. The severity of ethanol-induced hyperactivity was attenuated with choline treatment. In addition, subjects exposed to ethanol during the neonatal period committed a significantly greater number of perseverative-type errors on the reversal learning task compared to controls. Exposure to choline significantly reduced the number of ethanol-related errors. Importantly, these behavioral changes were not due to the acute effects of choline, but were related to long-lasting organizational effects of early choline supplementation. These data suggest that early dietary interventions may reduce the severity of fetal alcohol effects.

813 Preoperative concurrent chemoradiotherapy in non-small-cell lung cancer. Feasibility, toxicity and long-term results of a phase II study

Neonatal alcohol exposure impairs cognition and learning in adulthood and permanently damages the hippocampus. Wheel running (WR) improves hippocampus-associated learning and memory and increases the genesis and survival of newly generated neurons in the hippocampal dentate gyrus. WR significantly increases proliferation of newly generated dentate granule cells in alcohol-exposed (AE) and control rats on Postnatal Day (PD) 42 but only control rats show an increased number of surviving cells thirty days after WR (Helfer et al., 2009b). The present studies examined whether proliferation-promoting WR followed by survival-enhancing environmental complexity (EC) during adolescence could increase survival of new neurons in AE rats. On PD 4–9, pups were intubated with alcohol in a binge-like manner (5.25 g/kg/day, AE), were sham-intubated (SI), or were reared normally (suckle control, SC). On PD 30 animals were assigned to WR (PD 30–42) followed by EC (PD 42–72; WR/EC) or were socially housed (SH/SH) for the duration of the experiment. All animals were injected with 200 mg/kg bromodeoxyuridine (BrdU) on PD 41. In Experiment 1, survival of newly generated cells was significantly enhanced in the AE-WR/EC group in comparison with AE-SH/SH group. Experiment 2A examined trace eyeblink conditioning. In the SH/SH condition, AE impaired trace eyeblink conditioning relative to SI and SC controls. In the WR/EC condition, AE rats performed as well as controls. In Experiment 2B, the same intervention was examined using the context preexposure facilitation effect (CPFE); a hippocampus-dependent variant of contextual fear conditioning. Again, the WR/EC intervention reversed the deficit in conditioned fear to the context that was evident in the SH/SH condition. Post-weaning environmental manipulations promote cell survival and reverse learning deficits in rats that were exposed to alcohol during development. These manipulations may provide a basis for developing interventions that ameliorate learning impairments associated with human fetal alcohol spectrum disorders.

Alterations in Insulin-Like Growth Factor-I Signaling in Cardiomyocytes From Chronic Alcohol-Exposed Rats

Background Insulin-like growth factor-I (IGF-I) is a required cytokine for the development and maintenance of the cardiovascular system, and it may play a role in certain pathophysiological conditions. Methods Adult male rats were fed a liquid diet that contained 36% alcohol for 4 to 8 months, and their littermates served as isocaloric pair-fed controls, so we could examine IGF-I signaling in rat cardiomyocyte preparations. Results Recently, our laboratory reported that IGF-I activates protein kinase-C (PKC)-α and that PKC-α activity is required for IGF-I-dependent activation of Erk1/Erk2 and IGF-I-dependent protein synthesis. But in chronic alcohol-exposed rats, there is loss of PKC-α activation by IGF-I, represented as a reduction in PKC-α translocation to the membrane. In reverse transcription-polymerase chain reaction experiments, both the alcoholic and control animals expressed the IGF-I receptor (IGF-1R, α subunit) and IGF-I mRNAs in approximately equal amounts. However, in the alcoholic cardiomyocyte protein preparations, there was a higher basal level of IGF-1R autophosphorylation of its internal tyrosine kinase domain, and IGF-I-activated autophosphorylation was reduced in the alcoholic protein preparations. Previously, we have demonstrated that acute IGF-I exposure enhances the rate of Mn2+ influx through activated nitrendipine-sensitive cardiac Ca2+ channels, and that this enhancement of channel activity is PKC-dependent. Here, we report that in alcohol-exposed myocytes, IGF-I-induced augmentation of the cardiac Ca2+ channel activity was absent. IGF-I increased the rate of protein synthesis in the control animals by 56% as determined in protein synthesis experiments that measured the rate of 14C-L-phenylalanine incorporation over time, and this effect was blocked by preincubation with Gö6976, a specific inhibitor of PKC-α. However, in the alcohol-exposed cardiomyocytes, IGF-I did not increase the rate of protein synthesis. Conclusion These results suggest that IGF-I-dependent PKC-α activation and IGF-I-dependent protein synthesis are altered in the hearts of chronic alcohol-exposed rats. This may be the result of the IGF-1R being in a chronically activated state, and it may alter the normal function of PKC-α.

Alterations in Insulin‐Like Growth Factor‐I Signaling in Cardiomyocytes From Chronic Alcohol‐Exposed Rats

Background Insulin‐like growth factor‐I (IGF‐I) is a required cytokine for the development and maintenance of the cardiovascular system, and it may play a role in certain pathophysiological conditions.Methods Adult male rats were fed a liquid diet that contained 36% alcohol for 4 to 8 months, and their littermates served as isocaloric pair‐fed controls, so we could examine IGF‐I signaling in rat cardiomyocyte preparations.Results Recently, our laboratory reported that IGF‐I activates protein kinase‐C (PKC)‐α and that PKC‐α activity is required for IGF‐I‐dependent activation of Erk1/Erk2 and IGF‐I‐dependent protein synthesis. But in chronic alcohol‐exposed rats, there is loss of PKC‐α activation by IGF‐I, represented as a reduction in PKC‐α translocation to the membrane. In reverse transcription‐polymerase chain reaction experiments, both the alcoholic and control animals expressed the IGF‐I receptor (IGF‐1R, α subunit) and IGF‐I mRNAs in approximately equal amounts. However, in the alcoholic cardiomyocyte protein preparations, there was a higher basal level of IGF‐1R autophosphorylation of its internal tyrosine kinase domain, and IGF‐I‐activated autophosphorylation was reduced in the alcoholic protein preparations. Previously, we have demonstrated that acute IGF‐I exposure enhances the rate of Mn2+ influx through activated nitrendipine‐sensitive cardiac Ca2+ channels, and that this enhancement of channel activity is PKC‐dependent. Here, we report that in alcohol‐exposed myocytes, IGF‐I‐induced augmentation of the cardiac Ca2+ channel activity was absent. IGF‐I increased the rate of protein synthesis in the control animals by 56% as determined in protein synthesis experiments that measured the rate of 14C‐L‐phenylalanine incorporation over time, and this effect was blocked by preincubation with Gö6976, a specific inhibitor of PKC‐α. However, in the alcohol‐exposed cardiomyocytes, IGF‐I did not increase the rate of protein synthesis.Conclusion These results suggest that IGF‐I‐dependent PKC‐α activation and IGF‐I‐dependent protein synthesis are altered in the hearts of chronic alcohol‐exposed rats. This may be the result of the IGF‐1R being in a chronically activated state, and it may alter the normal function of PKC‐α.

Postnatal stress of early weaning exacerbates behavioral outcome in prenatal alcohol-exposed juvenile rats

Some of the behavioral deficits caused by prenatal or postnatal alcohol exposure have been demonstrated to be ameliorated by environmental manipulations such as handling or environmental enrichment. This experiment, in contrast, investigated whether behavioral deficits due to prenatal alcohol exposure could be exacerbated by a stressful experience, early weaning. Pregnant dams were given either a liquid diet with 35% of the calories derived from alcohol, a liquid diet without alcohol to control for any effects of the liquid diet administration, or ad libitum food and water. Half of each litter were weaned at 15 days of age (early weaning) and half were weaned at 21 days of age (normally weaned). Offspring were weighed, tested for activity in an open field at 18 days of age, and trained to find a hidden platform in the Morris water maze at 22–24 days of age. Alcohol-exposed subjects who were weaned early were more impaired in spatial navigation ability than any other group. Similarly, the combination of early weaning and prenatal alcohol exposure caused the slowest growth. All subjects exposed to alcohol, regardless of weaning condition, had greater latencies to find the platform than those from the two control groups. There was no synergistic effect of alcohol and stress on activity levels, but all early-weaned females were more active than normally weaned females; males did not show this effect. Thus, environmental stressors such as early weaning can compound detrimental symptoms of prenatal alcohol exposure. These results have implications for the understanding of the effects of the environment on neuronal plasticity.

Therapeutic effects of complex motor training on motor performance deficits induced by neonatal binge-like alcohol exposure in rats:: II. A quantitative stereological study of synaptic plasticity in female rat cerebellum

Twenty days of complex motor skill training in adult rats was previously demonstrated to rehabilitate motor performance deficits induced by binge alcohol exposure in neonatal rats. This follow-up study evaluated morphological plasticity in the paramedian lobule of the cerebellum (PML) using the same treatment and training regimens. On postnatal days (PD) 4–9, female Long–Evans rats were given either alcohol (Alcohol Exposure — AE, 4.5 g/kg/day via artificial rearing), exposure to gastrostomy control (GC) artificial rearing procedures, or reared normally as suckle controls (SC). After weaning, all rats were housed two to three per cage. At 180 days old, rats were randomly assigned either to a rehabilitation condition (RC: given 20 days of complex motor skill training), or to an inactive condition (IC: remained in their home cage). The AE rats were delayed in acquiring the training, but there were no group differences in performance over the last 2 weeks of training. Unbiased stereological techniques were used to evaluate PML volume, Purkinje cell and parallel fiber synapse density. Although total volume of PML was significantly reduced in the AE rats, complex motor skill training resulted in a significant increase in the PML molecular layer in all three postnatal treatment groups. The RC animals from the SC and AE groups had more parallel fiber synapses per Purkinje cell than corresponding IC animals. These data support the hypothesis that ‘rehabilitative’ motor training stimulates synaptogenesis in the PML, and that Purkinje neurons that survive the early postnatal alcohol insult are capable of substantial experience-induced plasticity.

Maternal alcohol and adrenalectomy:: Asynchrony of stress response and forced swim behavior

Fetal alcohol-exposed (FAE) rats exhibit heightened hormonal and behavioral stress responses, strikingly similar to those caused by exposure to elevated maternal corticosterone (CORT). Since alcohol increases maternal CORT, this study examined the effect of maternal adrenalectomy (ADX) on the CORT stress response and forced swim test (FST) behavior of the adult FAE offspring. Maternal ADX alone dramatically enhanced the CORT stress response of the offspring of pair-fed (PF) mothers but had no effect on the exaggerated CORT response to restraint stress observed in the FAE female. In contrast, maternal ADX reversed the increased immobility of FAE offspring in the FST of depressive behavior. These findings provide original evidence that stress hyper-reactivity and depressive behavior in the FAE offspring are mediated by separate developmental mechanisms.